Redox signaling at reperfusion is required for protection from ischemic preconditioning but not from a direct PKC activator

Abstract

Redox signaling prior to a lethal ischemic insult is an important step in triggering the protected state in ischemic preconditioning. When the preconditioned heart is reperfused a second sequence of signal transduction events, the mediator pathway, occurs which is believed to inhibit mitochondrial permeability transition pore formation that normally destroys mitochondria in much of the reperfused tissue. Prominent among the mediator pathway's events is activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase. Recently it was found that both activation of PKC and generation of reactive oxygen species (ROS) at the time of reperfusion are required for protection in preconditioned hearts. To establish their relative order we tested whether ROS formation at reperfusion is required in hearts protected by direct activation of PKC at reperfusion. Isolated rabbit hearts were exposed to 30 min of regional ischemia and 2 h of reperfusion. Preconditioned hearts received 5 min of global ischemia and 10 min of reperfusion prior to the index ischemia. Another group of preconditioned hearts was exposed to 300 microM of the ROS scavenger N-(2-mercaptopropionyl) glycine (MPG) for 20 min starting 5 min prior to reperfusion. Infarct size was measured by triphenyltetrazolium staining. Preconditioning reduced infarct size from 36% +/- 2% of the ischemic zone in control hearts to only 18 +/- 2%. MPG during early reperfusion completely blocked preconditioning's protection (33 +/- 3% infarction). MPG given in the same dose and schedule to non-preconditioned hearts had no effect on infarct size. In the last group phorbol 12-myristate 13-acetate (PMA) (0.05 nM) was given to non-preconditioned hearts from 1 min before to 5 min after reperfusion in addition to MPG administered as in the other groups. MPG did not block protection from an infusion of PMA as infarct size was only 9 +/- 2% of the risk zone. We conclude that while redox signaling during the first few minutes of reperfusion is an essential component of preconditioning's protective mechanism, this step occurs upstream of PKC activation.

Figure 1

Infarct size as a percentage of risk zone for individual hearts and group means with SEM. Abbreviations: IPC = ischemic preconditioning MPG = N-(2-mercaptopropionyl) glycine PMA = phorbol 12-myristate 13-acetate *p<0.001 vs. Control

Figure 2

Flowchart originally presented by Hausenloy et al (10) and our revision based on our new data